FaRIF at the Core of Strawberry Fruit Ripening: Deciphering its Targets and Interaction Networks

Ripening Inducing Factor (RIF) is a key NAC transcription factor regulating strawberry fruit ripening. Previous studies using RIF-RNAi and overexpression lines in Fragaria × ananassa and CRISPR knock-out lines in F. vesca have established the role of RIF in controlling ABA biosynthesis and signaling, cell wall remodeling, and secondary metabolism. In this study, we deciphered FaRIF's transcriptional regulatory network by combining ChIP-seq-based identification of its direct targets with an analysis of FaRIF-RNAi transcriptome data. These analyses revealed FaRIF's direct role in multiple aspects of strawberry fruit ripening, including the regulation of ripening-related transcription factors, phytohormone content and signaling, primary and secondary metabolism, and cell wall degradation. Additionally, using the TurboID-based proximity labeling approach, we have identified FaRIF interactors, including proteins involved in mRNA and protein homeostasis, as well as several NAC transcription factors. Among these, FaNAC034 was found to synergistically enhance FaRIF's transcriptional activity. This integrative analysis, combining transcriptome analysis, in vivo ChIP-seq, and proximity labeling, broadens our knowledge of FaRIF-mediated transcriptional networks and interaction partners, providing valuable insights into the molecular mechanisms underlying strawberry fruit ripening regulation by this transcription factor. Overall design: ChIP-seq of FaRIF from leaves of 35Spro:FaRIF-GFP plants, two biological replicates

成熟诱导因子（Ripening Inducing Factor，简称RIF）是调控草莓果实成熟的关键NAC转录因子。此前针对栽培草莓（Fragaria × ananassa）中RIF-RNAi及过表达株系、以及森林草莓（Fragaria vesca，简称F. vesca）中CRISPR敲除株系开展的相关研究，已明确RIF在调控脱落酸（Abscisic Acid，ABA）生物合成与信号通路、细胞壁重塑以及次生代谢过程中的功能。本研究通过将基于染色质免疫共沉淀测序（Chromatin Immunoprecipitation sequencing，ChIP-seq）的直接靶标鉴定结果，与FaRIF-RNAi转录组数据分析相结合，解析了FaRIF的转录调控网络。上述分析揭示，FaRIF可在草莓果实成熟的多个环节发挥直接调控作用，涵盖成熟相关转录因子、植物激素含量与信号通路、初生及次生代谢，以及细胞壁降解等方面的调控。此外，本研究借助基于TurboID的邻近标记（proximity labeling）技术，鉴定得到了FaRIF的互作蛋白，包括参与mRNA与蛋白质稳态调控的蛋白，以及多种NAC转录因子。在这些互作蛋白中，FaNAC034被证实可协同增强FaRIF的转录活性。本研究整合转录组分析、体内ChIP-seq以及邻近标记技术开展系统性分析，拓展了我们对FaRIF介导的转录调控网络及其互作伙伴的认知，为阐明该转录因子调控草莓果实成熟的分子机制提供了极具价值的参考依据。实验整体设计：对携带35S启动子（35S promoter, 35Spro）驱动的FaRIF-绿色荧光蛋白（Green Fluorescent Protein，GFP）融合基因的植株叶片进行FaRIF的ChIP-seq测序，设置2次生物学重复。